SOMATIC EMBRYOGENESIS ; 27 MARCH 15. 3.00 pm

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somatic embryogenesis in cereals crops

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WELCOME 3/27/2015 Deptt of Plant Biotechnology 1

In vitro Regeneration System for Indirect Somatic Embryogenesis in Cereal Crops.:

In vitro Regeneration System for Indirect Somatic Embryogenesis in Cereal Crops. 3/27/2015 Deptt of Plant Biotechnology 2 AVINASH SHARMA ID. No:- PALB 3235 Sr. M.Sc. (Plant Biotechnology)

CONTENTS:

CONTENTS Introduction. Importance of Somatic Embryogenesis. Types of Somatic Embryogenesis. Somatic Embryogenesis in Monocots and Dicots. Importance of Indirect Somatic Embryogenesis in Cereals. Indirect Somatic Embryogenesis in Cereal crops. Case Study. 3/27/2015 Deptt of Plant Biotechnology 3

Organogenesis and Somatic Embryogenesis::

Organogenesis and Somatic Embryogenesis: The development of adventitious organs or primordia from undifferentiated cell masses in tissue culture by the process of differentiation is called Organogenesis. It produces either root organ or shoot organ. It contain vascular bundles . 3/27/2015 Deptt of Plant Biotechnology 4

Stages of Organogenesis:

Stages of Organogenesis 3/27/2015 Deptt of Plant Biotechnology 5 1) Shoot tip in the medium 2) Shoot callus 3) Growth of stem 4) Root initiation 5) Hardening 6) Whole plant

Organogenesis in Cereal Crops::

Organogenesis in Cereal Crops: Crops Explants Results References 1) RICE cv “ Tainan 11” (TN11) and “Ai-Nan-Tsao 39” (ANT39) Immature seeds Callus induction:- MS+ 10µM 2,4-D (75%) in ANT 39; (88%) in TN11. Shoot Regeneration:- MS + 10µM NAA + 20µM KIN (80%) in ANT39; (0%) in TN11. Lee et al ., 2013. 2) Wheat cv (AS-2002; GA-2002 ). Immature embryo Callus induction:- MS+ 4mgl-¹ (92.75%) in AS-2002; (91.25%) in GA-2002 .Shoot Regeneration:- MS + 1.0 mgl­¹ (41.19%) in GA-2002. Mahmood et al ., 2012. 3/27/2015 Deptt of Plant Biotechnology 6

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3/27/2015 Deptt of Plant Biotechnology 7 Crops Explants Results References 3) Sorghum Mature embryo or Immature embryos Callus induction:- MS + pCPA 2.0mgl¯¹+ BAP 5.0mgl¯¹ (90%) in mature embryo, (95%) in immature embryo Regeneration:- MS + 2.0mgl¯¹+ IAA 0.5mgl¯¹ (39% ) in mature embryo and (48%) in immature embryo. Nirwan et al ., 2004. 4) Maize Genotypes (CML 427) Seeds Callus induction:- N6 + 5µM DICAMBA ( 86.67%) in CML 427; Shoot regeneration MS + 13.3µm BAP in CML 427. Matazu et al ., 2014.

Somatic Embryogenesis::

Somatic Embryogenesis: Somatic embryogenesis is a process by which somatic cells or tissues, including haploid cells develops into differentiated embryos and to regenerate plants. Stewart et al., (1958): First induced embryo through suspension culture in carrot. Reinert (1959): Produce embryo from callus in carrot through suspension culture. 3/27/2015 Deptt of Plant Biotechnology 8

Importance of Somatic Embryogenesis: :

Importance of Somatic Embryogenesis: Higher propagation rate. Suitable in Suspension culture. Artificial seed production. Labour savings . 3/27/2015 Deptt of Plant Biotechnology 9

Stages of Somatic Embryogenesis:-:

Stages of Somatic Embryogenesis:- 3/27/2015 Deptt of Plant Biotechnology 10 cytokinin

Somatic Embryogenesis in Cereal Crops::

Somatic Embryogenesis in Cereal Crops : Crops Explants Results References 1) Rice var. (MR 219) Seeds Callus induction:- 1mgl­¹ 2,4-D + 10mg/l + 10mgl­¹ NAA (78%) in MR 219; Embryo to Plants :- MS + 3mgl­¹+ 0.5mgl¯¹ NAA (88%) in MR219. Zuraida et al ., 2012. 2) Wheat loc . var. (GA-2002; Sahar) . Seeds Callus induction:- MS + 2mgl¯¹ 2,4-D (71%) in Sahar ; MS + 4mgl¯¹ (82.60%) in GA-2002; Shoot regeneration:- MS + 4mgl¯¹(88%) in GA-2002; MS + 4mgl¯¹ (73.51%) in Sahar. Munazir et al ., 2010. 3/27/2015 Deptt of Plant Biotechnology 11

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3/27/2015 Deptt of Plant Biotechnology 12 Crops Explants Results References 3) Maize cv. (Gaurav) Immature embryos Callus induction:- MS + 5.0mgl¯ 2,4-D (77.77%) in Gaurav; Shoot regeneration:- MS + 2mgl¯¹BAP (35-48%) in Gaurav. Joshi et al ., 2010. 4) Sorghum Genotypes (IS 3566, SPV 475) Seeds Callus induction :- MS + 2mgl¯¹ 2,4,5-T + 1.0 mgl¯¹ Zeatin (72%) in IS 3566; MS + 2.0mgl¯¹ 2,4,5-T + 1.0mgl¯¹ Zeatin (80%) in SPV 475. Regeneration :- MS + 2.5 mgl¯¹ TDZ (14 Shoots) in IS 3566; MS + 3mgl¯¹ TDZ (13 shoots) in SPV 475. Pola et al ., 2006.

Types of Somatic Embryogenesis:-:

Types of Somatic Embryogenesis:- Two types of somatic embryogenesis Direct somatic embryogenesis The embryos initiate directly from explants in the absence of callus formation. Embryos are formed due to PEDCs cell. Indirect somatic embryogenesis Callus from explants takes place from which embryos are developed. Embryos are formed due to IEDCs cells. 3/27/2015 Deptt of Plant Biotechnology 13

Direct Somatic Embryogenesis:

Direct Somatic Embryogenesis ffff 3/27/2015 Deptt of Plant Biotechnology 14 Yadav et al ., 2014

Indirect Somatic Embryogenesis:

Indirect Somatic Embryogenesis 3/27/2015 Deptt of Plant Biotechnology 15

Importance of Indirect Somatic Embryogenesis in Cereals::

Importance of Indirect Somatic Embryogenesis in Cereals: Indirect Somatic embryogenesis has high Plant regeneration capacity. Indirect Somatic embryogenesis reduces the breeding cycle. Indirect somatic embryogenesis are used in the crop improvement. Frequency of somaclonal variation is also very high in Indirect somatic embryogenesis Indirect somatic embryogenesis are better than the Direct somatic embryogenesis. 3/27/2015 Deptt of Plant Biotechnology 16

Somatic Embryogenesis in Monocots and Dicots::

Somatic Embryogenesis in Monocots and Dicots: Monocots Radicle protected by coleorrhiza and plumule coleoptiles in monocots. Cambial tissue are absent in monocots. Secondary growth are absent. Easy to culture in the media. Dicots Coleoptiles and Coleorrhiza are absent. Cambial tissue are present. Secondary growth are present. Difficult to grow in the media. 3/27/2015 Deptt of Plant Biotechnology 17

Indirect Somatic Embryogenesis in Cereals::

Indirect Somatic Embryogenesis in Cereals: 3/27/2015 Deptt of Plant Biotechnology 18 Crop: Japonica Rice cv. Kitaake seeds Callus induction media Regeneration Media: CHO source- maltose (40g/l) Hormones- NAA (0.2 mg/l) and BAP (3.0 mg/l), Agar (0-8, 1 and 1.2%) Hormones- 2,4-D and BAP (3.0 mg/l) Proline (0.6 g/l) and Phytagel (0.3%) Treatments: Either alone or in combination of Hormones, gelling agents, proline and maltose supplemented with basic MS media.

Results::

Results: Gelling agents Callus induction Regeneration 0.8% agar 52.47 (46.51) 28.33 (32.14) 1.0% agar 68.67 (55.94) 51.00 (45.56) 1.2% agar 70.67 (57.12) 38.00 (38.04) 0.3% Phytagel 92.00 (68.85) 60.54 (58.85) 0.8% agar + 0.2% Phytagel 87. 00 (68.85) 90.00 (71.83) 3/27/2015 Deptt of Plant Biotechnology 19 Callus induction medium: MS + 2,4-D (3.0mgl -1 ) + BAP (0.25mgl -1 ) + proline (0.6gl -1 ) + maltose (40gl -1 ) + agar (0.8, 1.0, 1.2%) , Phytagel (0.3%) or agar in combination with Phytagel. Regeneration medium : MS + BAP (3.0 mgl -1 ) + NAA (0.2mgl -1 ) + Phytagel (2gl -1 ) + agar (8gl -1 )

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3/27/2015 Deptt of Plant Biotechnology 20 Explants : Immature grains 5 Maize Inbred lines ( LM5, LM6, LM13, LM15 and LM16) Treatments: Different concentrations and combinations of Auxins and cytokinins. Auxins - Picloram (2.5, 5 and 10 mg/l), 2,4-D (3, 6 and 10 mg/ l) and NAA (5, 10 mg/l) Cytokinins- BAP (0.5 mg/l) and Kinetin (0.75 mg/l) Sucrose ( 60g/l), Agar (8gm/l) Observations were recorded on percent response of explants to callus induction (%) and Somatic embryogenesis(%)

Results:

Results 3/27/2015 Deptt of Plant Biotechnology 21 Table 1: Per cent of callus induction from immature embryos of five maize inbreds on different media compositions:-

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3/27/2015 Deptt of Plant Biotechnology 22 Table 2: Percent somatic embryogenesis induction in immature embryos of five maize inbred on different media compositions

CASE STUDY:

CASE STUDY 3/27/2015 Deptt of Plant Biotechnology 23

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Introduction:

Introduction Rice is the staple diet for two billion people world wide . It is feared that world population would be around 10 billion by 2050. Diminishing of cultivated land. Attack of pests and insects are responsible for decrease in production. There is a constant need to improve crops to overcome all these hazards. Somatic embryogenesis in rice has been reported culture of leaf tissue, root tissue, inflorescence and protoplast. 3/27/2015 Deptt of Plant Biotechnology 25

Materials and method:-:

Materials and method:- Explant collection:- Explant material for this research were rice seeds. Variety APMS-6B obtained from DRR (Hyderabad). Rice caryopses containing Scutellar region of embryo, were isolated by removing lemma and palea from the seeds . 3/27/2015 Deptt of Plant Biotechnology 26

Surface sterilization of Seeds:-:

Surface sterilization of Seeds:- Sterilization of rice caryopses using 70% alcohol for 3min. Followed by shaking in 30% Chlorox containing 2-3 drops of Tween-20 on an orbital shaker at 120 rpm for 20min. Explants were rinsed with sterile double sterilization water for 6 times. Cultured onto the medium with different treatment. 3/27/2015 Deptt of Plant Biotechnology 27

Preparation of Media:-:

Preparation of Media:- Two basic media used in this study:- First one was:- half MS (Murashige & Skoog, 1962) supplements with 500mg/l glutamine, 100 mg/l proline. Second one was:- N6 media supplemented with 500mg/l L -Glutamine. Both media were solidified with 0.2% agar. pH adjusted with 5.8. 3/27/2015 Deptt of Plant Biotechnology 28

Callus Induction Media:-:

Callus Induction Media:- Different concentrations of 2, 4-D [0.1, 1.5, 2.5,3.5 and 5 mgL-1 (w/v)] were used as the treatments for embryogenic callus induction. Media were kept in dark condition for 1 week, 25±2°C at room temperature. After 1 week transferred the cultures under 16 hrs lighting , provided by fluorescent bulbs with 15.75 µmolm-²s-¹ for eight weeks. 3/27/2015 Deptt of Plant Biotechnology 29

Somatic Embryo Germination Media:-:

Somatic Embryo Germination Media:- MS medium: BAP (0, 1, 2, 3, 4and 5 mg/l), NAA (0, 0.5, 1.0, 1.5, 2.5 and 4.0 mgL-1) Media were kept in the incubation room 25±2°C with 16 hrs of light provided by fluorescent bulbs and a light intensity of 16.75 µmolm-²s-¹ for eight weeks. Calculation: Callus induction frequency(%) Regeneration frequency(%). 3/27/2015 Deptt of Plant Biotechnology 30

Results:-:

Results:- After 3 days of culture callus started to grow from Scutellar embryo. Embryo derived callus subsequently started to enlarge and some yellowish to greenish nodules grew around explants after ten days. After 2 months of culture calli almost covered the explants surface. For callus induction MS medium supplemented with different concentration of 2,4-D(0, 1.0, 1.5, 2.5, 3.5 and 5 mg/l) was used in which 3.5 , 5 mg/l 2,4-D showed high callus induction percentage. It can be observed from Table 1 3/27/2015 Deptt of Plant Biotechnology 31

Table 1. Callus induction percent of rice: :

Table 1. Callus induction percent of rice: S. No Conc. Of 2,4-D (mgL-¹) Callus Induction Frequency % from rice 1. 0 No callus 2. 1.0 76±35 3. 1.5 80±40 4. 2.5 88±45 5. 3.5 95±30 6. 5.0 86±45 3/27/2015 Deptt of Plant Biotechnology 32 The result showed that the increased concentration of 2,4 –D more than 3.5 mgL-¹ decreased the callus formation percentage.

Contd:-:

Contd:- MS media supplemented with 0.8% agar, 70gm/l sucrose, 4gm/l Casein, 3mg/l BAP and 4 mg/l NAA was used for derived calli. 3 mg/l BAP concentration showed good results in Shoot induction, it can be observed from Table 3. 4 mg/l NAA concentration showed good results in Shoot induction, it can be observed from Table 2. 3/27/2015 Deptt of Plant Biotechnology 33

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S.No. Conc. Of NAA (mg/l) Shoot Induction % No. of Shoots 1. 0 31.33 2.6±0.48 2. 0.5 25.65 2.5± 0.64 3. 1 33.45 3.0± 0.54 4. 1.5 41.60 3.5± 0.64 5. 2.5 45.60 4.0± 0.59 6. 4.0 48.55 4.5± 0.60 Table 2. Effect of NAA PGRs in rice Table 3. Effect of BAP PGRs in rice S.No. Conc. Of BAP (mg/l) Shoot Induction % No. of Shoots 1. 0 30.33 2.0±0.87 2. 1 23.45 1.8±0.48 3. 2 31.85 2.2±0.16 4. 3 40.68 3.0±0.18 5. 4 38.67 2.5±0.64 6. 5 35.45 2.4±0.35 3/27/2015 Deptt of Plant Biotechnology 34

Contd:-:

Contd:- MS medium + different concentrations of NAA (0, 0.5, 1.0, 1.5, 2.0 mg/l) in combination with different concentrations of BAP (0, 1, 2, 3, 4, and 5 mg/l). Result showed that combination of 3mg/l BAP + 1.5 mg/l NAA showed highest result. Further combination increases cause the decrement of percent of Shoot induction. It can be observed from Table 4. 3/27/2015 Deptt of Plant Biotechnology 35

Table 4. Effect of BAP + NAA:

Table 4. Effect of BAP + NAA 3/27/2015 Deptt of Plant Biotechnology 36 S.No. BAP + NAA (mg/l) Shoot Induction % No. Of Shoots 1. 1 + 0.5 26.85 2.1± 0.63 2. 2 + 1.0 29.65 2.5 ±0.83 3. 3 + 1.5 39.60 3.5± 0.54 4. 4 + 2.0 35.45 3.2± 0.45 5. 5 + 4.0 30.40 3.0± 0.54

Immature embryos of APMS -6B seeds regenerate through Indirect Somatic Embryogenesis:

Immature embryos of APMS -6B seeds regenerate through Indirect Somatic Embryogenesis 3/27/2015 Deptt of Plant Biotechnology 37 Fig 1. Seed inoculation in MS medium Fig 2. Callus formation by 2, 4-D Fig 3. Shoot induction by differ. Conc. Of BAP and NAA Fig -4 Transplantation

Conclusion :

Conclusion Somatic embryogenesis is an efficient plant regeneration system under in vitro . It is potential useful tool for genetic transformation. Cross linking between hormone and transcription factors is likely to play an important part in SE. But mechanism of plant embryogenesis is unclear and comphrensive work in future is necessary to be studied with the interaction of various factors for entire picture of regulatory mechanism of embryogenesis to be transparent. 3/27/2015 Deptt of Plant Biotechnology 38

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